We propose that Nkx2.2 and NeuroD, two essential pancreatic transcription factors differentially regulate the development and differentiation of individual islet cell populations. Nkx2.2 and NeuroD are both required for the development of ( and ( cells in the mouse pancreatic islet. Consequently, mice lacking either of these factors die within days after birth due to neonatal diabetes. Nkx2.2-null mice completely lack insulin-producing ( cells and have a large decrease of glucagon-producing ( cells. In place of these cell types, the Nkx2.2 mutant islet is filled with cells producing the hormone ghrelin. Extensive analysis has indicated that Nkx2.2 is responsible for the initial specification of ( and ( cells. Alternatively, NeuroD appears to play a later role in islet cell differentiation as NeuroD-null mice display poor differentiation and extensive cell death among developing ( cells, disorganized ( cells, and subsequent failure to form islets. We have demonstrated that Nkx2.2 and NeuroD play critical roles in pancreatic cell differentiation, therefore understanding their precise functions in the islet will be essential for understanding of the molecular mechanisms that regulate pancreatic cell fate decisions. To define the specific roles of Nkx2.2 and NeuroD in each of the islet cell types and their respective roles in the function of the mature ( and ( cells, we have generated floxed-conditional knock-out mice for Nkx2.2 and NeuroD. We will use these mice to delete each gene in developing or mature ( or ( cells to determine their respective roles in each cell type. In addition, we will determine the precise molecular mechanisms of Nkx2.2 and NeuroD function in each individual cell type by introducing targeted mutations into each gene using the ES cell cassette exchange approach (M. Magnuson, see letter of collaboration). Finally, preliminary data suggests that Nkx2.2 and NeuroD have intersecting affects on ghrelin cell expression in the islets. We will determine the epistatic relationship between Nkx2.2 and NeuroD in the regulation of the ghrelin cells and the other islet cell types by generating Nkx2.2/NeuroD double null mice. Given that cell replacement therapy for Type I diabetes will heavily depend on our understanding of the factors involved in development of pancreatic beta cells, our research will have a significant impact on molecular and cellular approaches to generating functional endocrine cells for curing Type I diabetes. Specific Aim 1. Determine the spatio-temporal requirement of Nkx2.2 during pancreatic islet development by analyzing a conditional knockout of Nkx2.2. Specific Aim 2. Determine the spatio-temporal requirement of NeuroD during pancreatic development by anaylyzing a conditional knockout of NeuroD. Specific Aim 3. Determine the precise molecular mechanisms by which Nkx2.2 and NeuroD function in the pancreas using ES cell cassette exchange technology to generate mutations in each gene. Specific Aim 4. Determine the epistatic relationship between Nkx2.2 and NeuroD in the regulation of the islet cell type by creating Nkx2.2/NeuroD double null animals